Over 3 billion kg/yr of vinyl chloride (VC) are produced in the U.S. VC has been amply documented as a human carcinogen associated with lever haemoangiosarcoma and tumors of the brain and lungs. Although tumors are easily induced in rodents, and VC and its mutagenic metabolites, chloroethylene oxice (CEO) and chlorocetaldehyde (CAA) have been intensively studied, no mechanism for it tumorigenicity has emerged. Among the known products are four cyclic etheno bases. Our long-term objective is to understand the molecular mechanism of initiation by VC, related carcinogens and their common metabolites. The specific aims are directed towards studying the mispairing potential and repair mechanisms of the four cyclic adducts formed in DNA by vinyl chloride metabolites. The four etheno adducts are N2,3-ethenoG and its isomer 1,N3-ethenoG, as well as 1,N6-ethenoG and 3,N4-ethenoC. Study of repair of these adducts has become important with the recent finding by this laboratory of a human binding protein and a DNA glycosylate that excises ethenoA and ethenoC. One aim is to elucidate the mechanisms involved in this binding and removal. An additional aim is to determine whether the other etheno adducts, or those from related cyclic adducts, are also excised by this or other human repair enzymes. Concurrently, incorporated etheno adducts in different neighbor sequences in order to assess their mutagenic potential in vitro. The methods and approaches will involve chemical synthesis of precursor and preparation of oligonucleotides containing a single adduct. Use of such 32p-labeled probes will allow fidelity studies and detection of specific binding proteins using gel assay. Glycosylate action will be determined by HPLC using fluorescence or 3H labeled DNA. Proteins obtained by conventional purification and cloning will be used for obtaining repair enzymes for mechanistic studies. Formation and removal of the four etheno adducts will also be studied in cultured hepatocyte cells, by use of specific antibodies, 32P-postlabeling and HPLC analysis.